Many clinically important agents that are used to combat a wide variety of conditions are directed at inhibiting DNA replication. Inhibition of DNA replication can not only prevent proliferation of an infecting pathogen, but can also prevent proliferation of cancer cells. Typically, presently available agents inhibit DNA replication by interfering with DNA polymerases. A significant disadvantage of many of these agents is that DNA polymerases are often involved in cellular processes other than DNA replication, such as DNA repair. Another disadvantage that is sometimes observed is a lack of specificity for a particular target organism; such agents can inhibit desirable DNA replication of host cells, as well as replication in the target organism.
The shortcomings of many presently available agents for inhibiting DNA replication demonstrates the need for agents that inhibit DNA replication by a mechanism other than interference with DNA polymerase. One target of interest is primases, which are enzymes that catalyze the polymerization of an RNA oligonucleotide on a DNA template. These short oligonucleotides act as primers for DNA polymerases, which are not capable of initiating synthesis of a DNA strand de novo. Because of the critical functions played by primases, they provide promising targets for therapeutic intervention in, for example, pathogenic infection and cancer. For example, many large DNA viruses such as the herpes viruses have specific, targetable primases for which it would be desirable to develop specific inhibitors that do not interfere with primases of the infected organism.
The search for modulators of primases has been severely hampered by shortcomings with previously available assay methods. The standard assay for measuring primase activity uses solution-phase radiolabel incorporation and gel electrophoresis to monitor, directly or indirectly, the polymerization of nascent RNA oligonucleotides (see, Yoshida et al. (1985) J. BioChem. (Tokyo) 98: 427-433). Unfortunately, this assay is time-consuming, cumbersome and poorly suited to high-throughput drug screening. Detection of primase-synthesized primers in whole cell assays is difficult because the primers typically make up less than about one percent of the total cellular RNA (Tseng et al. (1977) Cell 12: 483-489; Palf et al. (1990) Biochemistry29: 3442-3450). A more recently developed assay method uses whole cell lysates and involves isolating nuclear matrices (Catapano et al. (1991) Cancer Res. 54: 1829-1835). This method, however, is also not suitable for high-throughput drug screening because a density gradient centrifugation step is required in order to purify the primase-synthesized primers. Thus, a need exists for new primase assays that are suitable for use in high-throughput screening methods. The present invention satisfies this and other needs.